Preparation of stock, calibration, and quality control standards
A stock solution of tofacitinib (1 mg/mL) was prepared by dissolving an accurately weighed amount of tofacitinib in methanol. The working standard solution (100 µg/mL) and calibration standards 50–15,000 ng/mL were prepared through serial dilution with methanol. The quality control (QC) standards were prepared from the standard stock at three concentration levels: low QC (250 ng/mL), medium QC (8000 ng/mL), high QC (12,000 ng/mL), and the lower limit of quantification (LLOQ) (50 ng/mL).
DoE methodology and optimization of analytical method
Using the above-executed trials, the organic phase and pH of phosphate buffer demonstrated a high impact on retention time and tailing factor. Therefore, to investigate the effect of organic phase composition and phosphate buffer pH on drug retention time and tailing factor, DoE methodology was applied [28]. A 32 factorial design consisting of 2 factors at 3 levels was considered for an experimental plan with Design-Expert 8.0 Stat-Ease Inc. Minneapolis, USA. The two independent variables % organic phase (X1), and pH of phosphate buffer (X2) with 3 levels (− 1 (3.5), 0 (4.5), + 1 (5.5)) were confiscated as the actual value. The retention time and tailing factor were considered as dependent variables as responses Y1 and Y2, respectively.
Validation of the developed method
The validation of the analytical method was performed for system suitability, linearity, range, detection limit, quantification limit, specificity, accuracy, precision, carryover effect, and robustness according to the ICH Q2 (R1) guideline (2005).
System suitability
System suitability test was preferred for chromatographic methods to ensure that the system is efficient to give reproducible results. The performance of the system was evaluated by injecting six replicates of 10 µg/mL concentration with optimized chromatographic conditions.
Linearity, limit of detection and quantification limit
Linearity was determined with a concentration range between 50 and 15,000 ng/mL with six calibration standards. The obtained data were fitted into linear regression analysis, and the calibration curve was plotted by the analyte peak area on the x-axis against the concentration of analyte on the y-axis. The detection limit (or) limit of detection (LOD) and quantification limit (or) limit of quantification (LOQ) were decided based on the signal-to-noise (S/N) ratio. Initially, in the system suitability test, the signal-to-noise ratio was obtained based on the detector response. The preferred S/N ratios were 3:1 and 10:1 for LOD and LOQ, respectively. LOD and LOQ were calculated based on the below mentioned formula [29]. From these, LLOQ has been determined and considered as the lowest standard of the calibration curve.
$${\mathrm{LOD}}\, {\text{and}}\,{\text{LOQ}}=\frac{{\mathrm{Concentration}}\,{\text{of}}\,{\text{standard}}}{\frac{S}{N }\,{\mathrm{value}} \,{\text{of}}\,{\text{standard}}}*{\mathrm{Desired}}\frac{S}{N}{\mathrm{value}}$$
Accuracy and precision
The measurement of the observed value's proximity to a given value is known as accuracy. Precision, on the other hand, relates to the closeness of measurement values to one another. The accuracy and precision of the quality control samples LQC, MQC, and HQC, as well as LLOQ, were determined in six replicates on intra- and inter-days. Accuracy was denoted as % bias and the precision as % relative standard deviation (RSD). The acceptance requirement for precision and accuracy of quality control samples according to regulatory criteria was ≤ ± 2% and ≤ ± 10% RSD, respectively [30].
Carryover effect
The carryover was assessed by analyzing successive samples (10 µg/mL, 12 µg/mL, and 15 µg/mL) of the linearity curve followed by a blank. Carryover acceptance criteria should not be higher than 20% of LLOQ.
Robustness
Robustness can be defined as the reproducibility potential of the developed method in the same laboratory conditions with slight modification in chromatographic conditions and different HPLC systems with specified conditions. Initially, the robustness of the developed analytical method was performed by changing the column oven temperature, to ± 5 °C, and mobile phase pH, to ± 0.5. Further, the optimized method was tested on another laboratory condition with the Shimadzu system (model number: LC 2010CHT).
Specificity
The specificity of the developed analytical method was studied in the presence of formulation excipients (lipids). A known concentration of tofacitinib within the linearity range was spiked into the lipids and analyzed using the developed HPLC method. The interference of lipids with the retention time of analyte and peak purity was observed.
Stability-indicating property of the developed method
The stability-indicating property of the developed analytical method was studied by exposing the tofacitinib solution to stress conditions as per ICH Q1A (R2) guidelines. The stress studies of tofacitinib solution were conducted under acidic hydrolysis, base hydrolysis, oxidation, and thermolytic conditions [31]. The acid hydrolysis and base hydrolysis were carried out by preparing the tofacitinib solution (200 µg/mL) using 0.5 M Hydrochloric acid and 0.5 M sodium hydroxide and kept for reaction on a water bath at 60 °C for 6 h. Similarly, for oxidative degradation, tofacitinib solution was prepared using 2% hydrogen peroxide and heated at 60 °C under reflux condition for 3 h. For thermal degradation, the tofacitinib solution was heated at 80 °C under reflux condition for 6 h. After subjecting tofacitinib solution (200 µg/mL) to the above-said stress conditions, a concentration of 10 µg/mL was prepared using methanol. The samples of acid and base were neutralized before dilution with methanol to protect the column. All the samples were filtered through a 0.2-µm filter before injecting it into HPLC analysis [32, 33]. The chromatogram of different stress conditions was recorded and compared with the normal condition. The retention time of different degradant peaks and drug peaks was identified and calculated the % degradation.
Applicability of the developed method for skin studies of tofacitinib and dermatokinetic assessment
The validated method for quantification of tofacitinib in the presence of skin tissue matrix was used to evaluate tofacitinib penetration through skin. Initially, skin tissue was homogenized using an Ultra-Turrax type homogenizer. Furthermore, the homogenized tissue was spiked with a predetermined concentration of tofacitinib solution (500 µg/mL) and centrifuged.. The supernatant (1 mL) was collected, and from this a concentration of 10 µg/mL was prepared. The samples were filtered through the 0.22-µ membrane filter before analysis and observed for peak specificity, and percent recovery was calculated [34]. In-house tofacitinib cream was prepared using stearic acid as base (0.5 mg/g) and applied topically (350 mg). After topical application, skin samples were collected for each time point at 2, 4, 6, 8, 12, 24 h. The collected skin samples were washed with phosphate buffer and gently wiped with the cotton. Tape stripping analysis was performed to separate the epidermis and dermis layers. The collected tapes and skin were soaked for 6 h in methanol. After 6 h, the samples were filtered through 0.25-µ filter and then analyzed at − 20 °C until analyzed as per validated method. Tofacitinib concentration in epidermis and dermis–time profiles were analyzed by non-compartmental model approach to determine t1/2, half-life; C0, tofacitinib concentration in epidermis and dermis at t = 0; AUC0–t, area under the curve from zero to the last measurable point; AUC0–∞, area under curve from time 0 extrapolated to infinity.
Applicability in characterization of nanoformulation
The validated analytical method was solicited for its use in the characterization of SLNs. The tofacitinib-loaded SLNs were prepared by the hot emulsification technique [35, 36]. The SLNs were prepared using Precirol as solid lipid and Poloxamer 407 as a surfactant as per the reported method. The prepared formulation was analyzed for its entrapment efficiency, in vitro drug release (pH 7.4), and stability studies.
Entrapment efficiency
The entrapment efficiency of prepared SLNs formulation was estimated by indirect method. In brief, the formulation was subjected to ultracentrifugation using Remi cooling centrifuge (Mumbai, India). The clear supernatant was diluted with the methanol and analyzed by the validated method.
$$\% \,{\mathrm{Entrapment}}\,{\text{Efficiency}}=1-\frac{{\mathrm{Free}}\,{\text{tofacitinib}}\,{\text{in}}\,{\text{nanocarrier}}\,{\text{dispersion}}}{\mathrm{Total}\,{\text{tofacitinib}}\,{\text{in}}\,{\text{nanocarrier}}\,{\text{dispersion}}}*100$$
In vitro drug release studies
The in vitro release of tofacitinib from SLNs dispersion was estimated using the dialysis bag technique. The study was performed using pH 7.4 phosphate buffer with 0.15% sodium lauryl sulfate. A known amount of formulation was transferred into the dialysis bag and maintained at 32 ± 0.5 °C. The samples were collected at regular intervals 1, 2, 4, 6 h and replaced with the fresh buffer to maintain sink condition. The tofacitinib concentration was assessed using an approved method after the samples were filtered through a 0.22-m filter.
Stability studies
Pharmaceutical formulation development involves stability at storage conditions to maintain drug activity. Thus, short-term stability of prepared SLNs formulation was carried out at room temperature and controlled condition for three months. The formulation was tested for entrapment efficiency after three months of storage period using the validated analytical method.